ABSTRACT ? PROJECT 2 This preclinical project uses canine models of nonmalignant blood disorders to develop conditioning regimens for allogeneic hematopoietic cell transplantation (HCT; Project 1) and gene therapy (Project 3) that have minimal toxicity, are safe, avert the short- and long-term toxicities such as growth failure, infertility, and late cancers, while at the same time achieving high-level donor multi-lineage engraftment. We seek to accomplish these goals with monoclonal antibody (MAb) targeted radioimmunotherapy (RIT) using a powerful alpha- emitting radionuclide, astatine-211 (211At). Alpha-emitters are characterized by efficient cell killing due to their very short path length (40-70 ?m) and high linear energy transfer. Targeted irradiation will avoid the short- and long-term toxicities of systemic conditioning regimens used up to now. Specific Aim 1: Develop conditioning regimens for DLA-identical and DLA-haploidentical HCT using 211At-labeled anti-CD45 MAb in a dog model of red blood cell disorders. Here we intend to overcome engraftment problems and toxicities of conventional conditioning by using targeted irradiation with an 211At-labeled panhematopoietic anti-CD45 MAb. Pyruvate kinase (PK) deficient dogs serve as a model of hemoglobinopathy. We will optimize 211At dosing for both DLA-identical and DLA-haploidentical HCT. Specific Aim 2: Develop conditioning regimens for DLA-haploidentical HCT using 211At-labeled anti- CD45 MAb in a dog model of X-linked severe combined immunodeficiency (SCID-X1). HCT with minimal or no conditioning often results in deficient B-cell engraftment and function in patients with SCID-X1. A modest degree of stable donor marrow engraftment is also required for long-term cure. We propose that a regimen incorporating 211At-labeled anti-CD45 MAb will achieve multi-lineage engraftment, with minimal toxicity in patients with SCID-X1. Specific Aim 3: Evaluate the use of 211At-labeled anti-CD45 MAb to overcome transfusion-induced sensitization and subsequent graft rejection in a well-established DLA-identical HCT model. Studies will be conducted to determine the optimal doses of 211At-labeled anti-CD45 MAb that are needed to target the immune cells responsible for graft rejection and consistently establish engraftment of donor hematopoietic cells with minimal toxicity. Specific Aim 4: Ex vivo expanded DLA-identical hematopoietic stem cells to facilitate engraftment after nonmyeloablative conditioning with 211At. There is no benefit in the development of graft versus host disease in patients with nonmalignant disorders. This aim will explore the ability to use RIT to engraft very high numbers of expanded stem cells (developed in Project 3) as a model of T-cell-depleted allogeneic HCT.